Prebiotic and preservative uses of oil emulsified probiotic encapsulations

ABSTRACT

An encapsulation system is provided comprising nitrogen-purge, instant bonding encapsulation method. Specifically, the encapsulation system comprises a composition, a two-piece capsule comprising a capsule cap and a capsule body; a gas to purge oxygen from the composition within the capsule; and a sealing solution to seal the capsule cap to the capsule body. Associated methods for encapsulating compositions using the encapsulation system are also provided.

RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 11/734,176 filed Apr. 11, 2007, which is a divisional of U.S.patent application Ser. No. 10/672,668 filed Sep. 26, 2003, now U.S.Pat. No. 7,214,370 which claims priority to U.S. provisional patentapplication 60/414,083 filed Sep. 26, 2002, now expired. U.S. patentapplication Ser. No. 11/734,176 is also a continuation-in-part of U.S.patent application Ser. No. 10/021,871, filed Dec. 17, 2001, now U.S.Pat. No. 6,797,266. The contents of each of these applications areincorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention is directed at probiotic compositions and methodsfor making same. More specifically, the present invention is directed atprobiotic compositions derived from lactic acid bacteria that have beenemulsified in prebiotic edible oils and packaged in an anaerobicencapsulation system.

BACKGROUND OF THE INVENTION

Probiotics are microbial-based dietary adjuvants that beneficiallyaffect the host physiology by modulating mucosal and systemic immunity,as well as improving nutritional and microbial balance in the intestinaltract [Naidu A S et al. Probiotic spectra of lactic acid bacteria (LAB).Crit. Rev. Food Sci. Nutr. 39:3-126, 1999]. Lactic acid bacteria (LAB)are indigenous probiotic microflora of mammalian gastrointestinal tractthat play an important role in the host microecology and have beencredited with an impressive list of therapeutic and prophylacticproperties [Naidu A S, Clemens R A Probiotics, p. 431-462. In AS Naidu(ed.), Natural Food Antimicrobial Systems. CRC Press, Boca Raton, Fla.,2000]. These therapeutic and prophylactic properties include, but notlimited to the maintenance of microbial ecology of the gut,physiological, immuno-modulatory and antimicrobial effects [Gibson G Ret al., Probiotics and intestinal infections, p. 10-39. In R. Fuller(ed.), Probiotics 2: Applications and practical aspects. Chapman andHall, London, UK, 1997]. Other LAB associated attributes include enzymerelease into the intestinal lumen that act synergistic with LAB adhesionto alleviate symptoms of intestinal malabsorption. Furthermore, theLAB-released enzymes help regulate intestinal pH that results inincreased aromatic amino acid degradation [Mitsuoka T Taxonomy andecology of bifidobacteria. Bifidobacteria Microflora 3:11, 1984]. LABhave also demonstrated the ability to significantly reduce sulfide andammonia containing compounds in animal fecal waste and thus reduce theodor and toxicity associated with animal excrements [Naidu A S et al.,Reduction of sulfide, ammonia compounds and adhesion properties ofLactobacillus casei strain KE99 in vitro. Curr. Microbiol. 44:196-205,2002].

However, the greatest potential for LAB to improve life quality for manand domestic animals lies in their in vivo probiotic applications. Inorder for LAB to exhibit beneficial probiotic effects in vivo, theorganisms must survive for extended time periods in the gut. Therefore,it is critical that probiotic LAB strains be selected that possessqualities that prevent their rapid removal by gut contraction [HavenaarR et al., Selection of strains for probiotic use, p. 209-224. In R.Fuller (ed.), Probiotics, the scientific basis. Chapman and Hall,London, UK, 1992]. Effective probiotic bacteria should be able tosurvive gastric conditions and colonize the intestine, at leasttemporarily, by adhering to the intestinal epithelia [Conway P.Selection criteria for probiotic microorganisms. Asia Pacific J. Clin.Nutr 5:10-14, 1996].

Furthermore, in addition to increasing in vivo viability andgastrointestinal tract life span, prolonged shelf life at roomtemperature remains a commercial challenge. Lactic acid bacilligenerally require an effective delivery system that retainsprobio-functional activities (i.e. gut adhesion/retention, production ofbacteriocins/enzymes) after their revival [Salminen S et al., Clinicaluses of probiotics for stabilizing the gut mucosal barrier: successfulstrains and future challenges. Antonie Van Leeuwenhoek 70:347-3581,1996]. Though freeze-drying is an effective process for preservation anddelivery of probiotics, several physico-chemical factors such ashumidity, aeration (oxygen availability) and temperature couldcompromise the cell viability, thereby the shelf life.

One potential additive class that may increase both in vivo life spanand storage shelf-life is prebiotics. Prebiotics are non-digestible, orpartially digestible, food ingredients that beneficially affect the hostby selectively simulating the growth and/or activity of one or a limitednumber of bacterial species and thus in effect improve host health.[Gibson G R, Roberfroid M B. Dietary modulation of the human colonicmicrobiota: Introducing the concept of prebiotics. J. Nutr. 125:1401-12,1995]. Intake of prebiotics can beneficially modulate probiotic LAB.Non-digestible oligosaccharides such as dietary fiber in general, andfructo-oligosaccharides (FOS) in particular, are well known prebiotics[Roberfroid M B. Health benefits of non-digestible oligosaccharides.Adv. Exp. Med. Biol. 427:211-9, 1997]. By combining the rationale ofprobiotics and prebiotics, the concept of ‘synbiotics’ is proposed tocharacterize some colonic foods with interesting nutritional propertiesin combination with health-enhancing functional food ingredients [FullerR, Gibson G R Modification of the intestinal microflora using probioticsand prebiotics. Scand. J. Gastroenterol. Suppl. 222:28-31, 1997].

Essential oils are known as biological preservatives due to their lowwater activity and limited air diffusion. Several essential oils alsoknown to provide various nutraceutical benefits including antioxidant,antimicrobial, antitumor, and immune-modulatory activities. However, theprebiotic effects of essential oils on probiotic LAB are heretoforeunknown. Therefore, there remains a need to enhance probiotic activity,in vivo viability and shelf life of probiotic compositions includingLAB. One potential solution is the application of prebiotics incombination with advanced packaging methods.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide enhanced probioticcompositions having increased viability, in vivo life span and increasedshelf life.

It is another object of the present invention to provide a prebioticcomposition in combination with a new strain of lactobacillus havingincreased viability, in vivo life span and increased shelf life.

It is yet another object of the present invention to preserve theprobiotic qualities of Lactobacillus casei KE01 including, but are notlimited the use of the prebiotic oil compositions of the presentinvention in combination with by an encapsulation process using a novelNitrogen-Purge, Instant-Bonding (NPIB) system.

The present invention fulfills these and other objects by providing anew strain of Lactobacillus casei designated KE01 that possessesscientifically proven probiotic properties including demonstrated invivo anti-enteric pathogen activity. Moreover, the present inventionprovides dietary supplements and pharmaceutical preparations composed ofL. casei strain KE01 that are formulated in a prebiotic composition ofedible oils that provided long term protection to the organism and helpmaintain its proven probiotic properties and increased in vivo life spanand shelf life.

There is a need for new probiotic formulations that can be used to treatand prevent enteric-pathogen infections and help maintain the health andvitality of humans and livestock. Recently, the Federal Food and DrugAdministration (FDA) has intensified its campaign against the overprescription and clinical abuse of antibiotics. The excessive use ofantibiotics has increased in the number of human and animal pathogensthat are resistant to first-line antibiotics resulting in an increase ininfections that do not respond to conventional antimicrobial therapies.Moreover, the prophylactic use of antibiotics in animal feed hasresulted in an alarming increase in livestock intestinal infectionsresulting in diminished herd size and animal weight due to nutrientmalabsorption. Consequently, the number of healthy animals suitable forhuman consumption has dropped, and those that do survive long enough toreach market have significantly lower weights and consequently reducedmeat quality.

One means of preventing the rapid spread of drug resistant entericpathogens in humans and livestock is to significantly reduce antibioticuse. However, the spread of communicable diseases including entericinfections is inevitable due to over crowding of farms and cities.Consequently, before prophylactic antibiotic use can be completelydiscontinued a suitable antimicrobial alternative must be available.Recent studies have indicated that the use of foodstuffs and dietarysupplements containing specific strains of probiotic microorganisms canhelp prevent, and in many cases actually cure, enteric pathogendiseases. However, many of the probiotic formulas currently marketedrely on organisms including Lactobacillus spp and Bifidobacteria sp (andother genera) that have not been subjected to scientific scrutiny usingapproved methods for assessing probiotic efficacy. Consequently, toomany of the “probiotic” formulas currently available lack proven in vivoanti-enteric pathogen activity. Moreover, many of the clinicallyeffective probiotic formulations commercially available are not stableupon storage and therefore do not deliver effective amounts of viableprobiotic bacteria to the user. The present inventors have tested manycommercially available preparations and found microbial viability wellbelow stated concentrations and in many cases the present inventor hasfound that these commercial preparations did not contain any viablebacteria.

The present inventors have developed methods for preparing and packaginga new strain of L. casei, designated KE01. This new strain of L. caseiwas originally from a traditional fermented yogurt-like Asian dairyproduct by the present inventor. Subsequently, the present inventorcharacterized the isolate and the strain deposited with the AmericanType Culture Collection (ATCC, MD, USA)). Lactobacillus casei strainKE01 has been given the ATCC depository number PTA 3945. Moreover, thepresent inventors have developed preparations and packaging systems thatmaintain L. casei KE01 viability such that a clinically effective doseof viable probiotic microorganisms reaches the host.

The present invention provides a L. casei strain (KE01) that interfereswith bacterial adherence (microbial interference) of enteric pathogenssuch as, but not limited to enteropathogenic and enterotoxigenic E.coli, Helicobacter pylori, Campylobacter jejuni, S. typhimurium, and S.enteritidis to a variety of mammalian cell types. Moreover, theLactobacillus of the present invention can also competitively exclude(competitive exclusion) these, and other bacterial pathogens, frombinding to many mammalian cells. The beneficial properties associatedwith the novel Lactobacillus strain of the present invention haveresulted in improved probiotic dietary supplements that support generalhuman and animal health. Moreover, the present invention can be used toprovide prophylactics, therapeutics and palliatives (collectivelyreferred to herein as “probiotics”) for conditions such as, but notlimited to, traveler's diarrhea, gastrointestinal infections, hemolyticuremic syndrome, and gastric ulcers.

Additional novel features and qualities of this new L. casei strain KE01include, but are not limited to, L. casei KE01's ability to reducesulfide concentrations by a factor exceeding 300 ppm within 48 hourswhen exposed to a growth medium containing approximately 2000 ppm ofsulfides and the demonstration of avid binding to sub-epithelialmatrices including Bio-coat™ (Collagen type-I, Collagen type IV,laminin, and fibronectin), Matrigel™ and Caco-2 cell monolayers. Mostimportantly, a reconstituted, freeze-dried preparation of the L. caseiof the present invention has been shown to effectively detachcollagen-adherent E. coli.

The methods used to maintain the viability of the L. casei of thepresent invention and preserve probiotic qualities include, but are notlimited the use of the prebiotic oil compositions of the presentinvention in combination with by an anaerobic encapsulation processusing a novel Nitrogen-Purge, Instant-Bonding (NPIB) system.

These and other beneficial probiotic properties of the new strain ofLactobacillus will be further evident by the following, non-limiting,detailed description of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the genomic fingerprint of Lactobacillus casei strainKE01 on 1% agarose gel compared to 12 different Lactobacillus typestrains based on Randomly Amplified Polymorphic DNA (RAPD) assay.

FIG. 2 depicts the phylogenic dendogram deduced from genomicfingerprinting and the relatedness of Lactobacillus casei strain KE01with other species of Lactobacillus type strains.

FIG. 3 depicts the standardized real-time PCR used to quantifylactobacillus content of the present invention using KE01 specificprimers.

FIG. 4. depicts sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) results using the KE01 primers of the presentinvention.

FIG. 5 depicts the process for making the anaerobic encapsulation systemusing NPIB in accordance with the teachings of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Lactic acid bacteria (LAB) are indigenous microflora of mammaliangastrointestinal tract that play an important role in the hostmicroecology and have been credited with an impressive list oftherapeutic and prophylactic properties. These therapeutic andprophylactic properties include, but not limited to the maintenance ofmicrobial ecology of the gut, physiological, immuno-modulatory andantimicrobial effects. Other LAB associated attributes include enzymerelease into the intestinal lumen that act synergistically with LABadhesion to alleviate symptoms of intestinal malabsorption. Furthermore,the LAB enzymes help regulate intestinal pH which results in increasedaromatic amino acid degradation. [Fuller R. Probiotic foods—current useand future developments. IFI NR 3:23-6, 1993; Mitsuoka T Taxonomy andecology of Bifidobacteria. Bifidobacteria Microflora 3:11, 1984; GibsonG R et al., Probiotics and intestinal infections, p. 10-39. In R. Fuller(ed.), Probiotics 2: Applications and practical aspects. Chapman andHall, London, U.K. 1997; Naidu A S et al., Probiotic spectra of lacticacid bacteria (LAB). Crit Rev Food Sci Nutr 39:3-126. 1999; Naidu A S,Clemens R A. Probiotics, p. 431-462. In AS Naidu (ed.), Natural FoodAntimicrobial Systems. CRC Press, Boca Raton, Fla., 2000].

Lactic acid bacteria have also demonstrated the ability to significantlyreduce sulfide and ammonia containing compounds in animal fecal wasteand thus reduce the odor and toxicity associated with animal excrements.This ex vivo LAB application is becoming increasingly more important asagro-businesses expand and as communities continue their seemingly neverending encroachment into previously unoccupied rural areas. For example,LAB has been demonstrated to eliminate offensive odors and reducehydrogen sulfide production associated with hatchery waste when cockerelchicks and shell waste are blended with a mixture containing 15%carbohydrate and LAB. Moreover, LAB compositions have demonstratedefficacy in diminishing the Escherichia coli and Salmonella content ofhatchery waste to negligible levels.

Additionally, the odor and viscosity of poultry offals such asbroiler-processing waste is significantly reduced by L. acidophilusmediated lactic acid fermentation. Furthermore, preparations containingLAB have been reported to accelerate the breakdown of hard-to-degradecarbohydrates and decrease the ammonia production by porcine cecalbacteria. Finally, ex vivo L. casei FG1 and L. plantarum silagefermentation significantly reduces ammonia levels by inhibitingurea-splitting organisms. [Deshmukh A C, Patterson P H. Preservation ofhatchery wastes by lactic acid fermentation. 1. Laboratory scalefermentation. Poult Sci 76:1212-19, 1997; Russell S M et al., Lacticacid fermentation of broiler processing waste: physical properties andchemical analyses. Poult Sci 71:765-70, 1992; Tibbetts G W et al.,Poultry offal ensiled with Lactobacillus acidophilus for growing andfinishing swine diets. J Anim Sci 64:182-90, 1987; Sakata T et al.,Probiotic preparations dose-dependently increase net production rates oforganic acids and decrease that of ammonia by pig cecal bacteria inbatch culture. Dig Dis Sci 44:1485-93, 1999; Cai Y et al., Effect ofapplying lactic acid bacteria isolated from forage crops on fermentationcharacteristics, aerobic deterioration of silage. J Dairy Sci 82:520-6,1999; Modler H W et al., Bifidobacteria and bifidogenic factors. CanInst Food Sci Tech 23:29-41, 1990].

However, the greatest potential for LAB to improve life quality for manand domestic animals lies in LAB in vivo probiotic applications. Inorder for LAB to exhibit beneficial probiotic effects in vivo, theorganisms must survive for extended time periods in the gastrointestinaltract. Therefore, it is critical that probiotic LAB strains be selectedthat possess qualities that prevent their rapid removal by gutcontraction. Effective probiotic bacteria must able to survive gastricconditions and colonize the intestine, at least temporarily, by adheringto the intestinal epithelium. Consequently, LAB that demonstrate anenhanced ability to adhere to mucosal surfaces, and therefore possessimproved bacterial maintenance and prolonged gastrointestinal tractresidence times, have a competitive advantage over LAB that do not.[Salminen S et al., Clinical uses of probiotics for stabilizing the gutmucosal barrier: successful strains and future challenges. Antonie VanLeeuwenhoek 70:347-58, 1996; Conway P. Selection criteria for probioticmicroorganisms. Asia Pacific J Clin Nutr 5:10-14, 1996; Havenaar R etal., Selection of strains for probiotic use, p. 209-224. In R. Fuller(ed.), Probiotics, the scientific basis. Chapman and Hall, London, U.K.,1992].

Lactobacillus can successfully colonize the mammalian gastrointestinaltract through a number of different mechanisms. For example, somebacterial species bind to various sub-epithelial matrix proteins andspecific receptors on the intestinal mucosa. Other species may adhere tomammalian intestinal cells via mechanisms that involve differentcombinations of carbohydrate and protein factors on the bacteria andhost eukaryotic cell surfaces. However, regardless of the mechanism(s)of attachment, it is the ability of LAB to successfully colonize thehuman gastrointestinal tract that provides LAB with probiotic qualities.[Greene J D, Klaenhammer T R Factors involved in adherence oflactobacilli to human Caco-2 cells. Appl Environ Microbiol 60:4487-94,1994; Sarem F et al., Comparison of the adherence of three Lactobacillusstrains to Caco-2 and Int-407 human intestinal cell lines. Lett ApplMicrobiol 22:439-42, 1996; Naidu A S et al., Particle agglutinationassays for rapid detection of fibronectin, fibrinogen, and collagenreceptors on Staphylococcus aureus. J Clin Microbiol 26:1549-54, 1988;Wadstrom T et al., Surface properties of lactobacilli isolated from thesmall intestines of pigs. J Appl Bacteriol 62:513-20, 1987; Bernet M Fet al., Lactobacillus acidophilus LA 1 binds to cultured humanintestinal cell lines and inhibits cell attachment, invasion byentero-virulent bacteria. Gut 35:483-9, 1994; Jin L Z et al., Effect ofadherent Lactobacillus spp. on in vitro adherence of salmonellae to theintestinal epithelial cells of chicken. J Appl Bacteriol 81:201-6, 1996;Reid G et al., Influence of lactobacilli on the adhesion ofStaphylococcus aureus and Candida albicans to fibers and epithelialcells. J Ind Microbiol 15:248-53, 1995].

Generally speaking probiotic bacteria exert their beneficial effects bydisplacing invasive or toxigenic pathogenic enteric bacteria (entericpathogens) from the intestinal mucosa through a competitive bindingprocess. Enteric pathogens such as, but not limited to, enteropathogenicEscherichia coli (EPEC), enterotoxigeneic E. coli (ETEC), Salmonellaenteriditis, Yersina pseudotuberculosis and Listeria monocytogenes mustbe able to successively colonize an animal's intestinal tract in orderto cause disease.

Probiotic compositions exert optimal beneficial qualities when thepercentage of viable probiotic bacteria is high. However, maintainingbacteria viability constitutes a significant challenge to the probioticindustry. Consequently, most probiotic compositions have a relativelyshort shelf life or are used with percent viabilities that aresuboptimal. Many probiotic compositions, including those of the presentinvention are made using selected stains of (or combination of) bacteriaincluding Lactobacillus acidophilus, L. amylovorus, L. brevis, L.bulgaricus, L. casei spp. casei, L. casei spp. rhamnosus, L. crispatus,L. delbrueckii ssp. lactis, L. fermentum, L. helvaticus, L. johnsonii,L. paracasei, L. pentosus, L. plantarum, L. reuteri, and L. sake: thegenus Bifidobacterium including: B. animalis, B. bifidum, B. breve, B.infantis, and B. longum: the genus Pediococcus including: P.acidilactici: the genus Propionibacterium including: P. acidipropionici,P. freudenreichii, P. jensenii, and P. theonii: and the genusStreptococcus including: S. cremoris, S. lactis, and S. thermophilus(collectively referred to herein after as lactic acid bacteria, or LAB).

Presently, probiotic compositions are produced using cultured,concentrated LAB that are dried or lyophilized and then mixed withstabilizing ingredients such proteins and sugars including, but notlimited raffinose, soybean oligosaccharides, fructooligosaccharides,galactooligosaccharides, galactosyl lactose and palatinose, lactulose,lactitol, xylitol, sorbitol, mannitol, trehalose, glucose, sucrose,fructose, maltose, milk, milk powders, whey, whey protein concentrates,casein, casein hydrolysates, lactoferrin, lactoperoxidase,lactoglobulins, glycomacropeptides, lacto-saccharides, and lacto-lipids.These probiotic mixtures are milled into small granule or fine powdersand then sealed in various pharmaceutically acceptable forms andpackaged. Alternatively, liquid preparations are provided that must bestored at refrigerator temperatures. However, regardless of the form,whether dried or liquid, LAB viability begins to drop dramatically soonafter shipping to retail outlets and end users.

The present inventors have surprisingly discovered that certain edibleoils not only protect LAB viability better than prior art stabilizingingredients such proteins and sugars, actually enhance viabilityresulting in a prebiotic effect. As previously discussed, prebiotics arenon- or partially digestible food ingredients that beneficially affectthe host by selectively simulating the growth and/or activity of one ora limited number of bacterial species in the colon, and thus in effectimprove host health. One embodiment of the present invention is adietary supplement comprising approximately from 10⁵ to 10¹¹ colonyforming units of viable LAB per mL of prebiotic edible oil. However, itis understood that the probiotic compositions of the present inventionare not limited by this range and may in fact comprises fromapproximately 1 to >10¹² colony forming units of viable lactobacilli permL of prebiotic edible oil.

In one embodiment of the present invention the LAB Lactobacillus caseistrain KE01 having the American Type Culture Collection (ATCC) accessionnumber PTA 3945. In one embodiment of the present invention theprebiotic/probiotic composition comprises a hard two piece capsulefilled with a lactobacillus composition suspended in at least one edibleoil. In this embodiment the hard two piece capsule has been purged withan inert gas upon filling and sealed to assure an oxygen-freeenvironment, as described more fully below. The hard two piece capsulecan be derived from either vegetables, animal gelatin or synthetic andnatural polymers.

Throughout this specification the present invention may be referred toas a probiotic composition, a lactobacillus containing composition, adietary supplement, or a probiotic/prebiotic composition. All of theseaforementioned terms mean a composition, regardless of form or thepresence or absence of other ingredients, that contains viable andor/non-viable LAB and at least one prebiotic, edible oil. In oneembodiment the LAB is Lactobacillus casei strain KE01 having ATCCaccession number PTA 3945 or it genetic equivalent as determined usingthe methods detailed herein.

In one embodiment of the present invention an animal is provided with asingle dose containing from approximately 10⁵ to 10¹¹ lactobacilli pergram of probiotic composition. The total amount consumed will depend onthe individual needs of the animal and the weight and size of theanimal. The preferred dosage for any given application can be easilydetermined by titration. Titration is accomplished by preparing a seriesof standard weight doses each containing from approximately 10⁵ to 10¹¹lactobacilli per gram. A series of doses are administered beginning at0.5 grams and continuing up to a logical endpoint determined by the sizeof the animal and the dose form. The appropriate dose is reached whenthe minimal amount of lactobacilli composition required to achieve thedesired results is administered. The appropriate dose is also known tothose skilled in the art as an “effective amount” of the probioticcompositions of the present invention.

For example, if it is desired to reduce the symptoms associated withirritable bowel syndrome in an animal, one measured dose as describedabove is administered daily, escalating the dose each successive day in0.5 grams increments until symptoms subside. In one embodiment of thepresent invention the preferred dose is between approximately 10³ to 10⁸viable lactobacilli per kilogram of body weight (the weight of theanimal recipient) per day. This equates to approximately 10 billionviable LAB per day for the average healthy adult human. Byextrapolation, it is a simple matter to calculate the approximate doseappropriate for any animal of any weight. It is understood that this isa non-limiting example that can be varied as appropriate by personshaving skill in the art of prescribing probiotic compositions or byusing the titration method provided above.

The probiotic compositions of the present invention can be administeredto any animal in need of thereof including, but not limited to mammals,birds, reptiles and fish. Typical applications include administering theprobiotic compositions of the present invention to humans, horses, swine(pigs), cows, sheep, dogs, cats, rabbits, chickens, turkeys, pheasants,quail, parakeets, parrots, and other wild and domesticated animals.

Specifically, the probiotic compositions of the present invention can beused to inhibit or treat enteric pathogen-associated diseases whenadministered to an animal in need thereof using the methods described inthe present specification. Enteric pathogen diseases include thosediseases caused by pathogens such as diarrhea, irritable bowel syndromeand intestinal hemorrhages. Examples of enteric pathogens associatedwith these diseases include, but not limited to enteropathogenicEscherichia coli (EPEC), enterotoxigeneic E. coli (ETEC), Salmonellaenteriditis, Yersina pseudotuberculosis and Listeria monocytogenes. Itis theorized by the present inventor, and not offered as a limitation,that the inhibition and treatment of the enteric pathogen diseases isaccomplished by the probiotic composition of the present inventionthrough a competitive binding process. That is, the probioticlactobacilli of the present invention compete with enteric pathogens forbinding sites on the intestinal mucosa. Because the probioticlactobacilli of the present invention have a higher affinity and avidityfor these binding sites than the enteric pathogens, the probioticlactobacilli of the present invention displace the enteric pathogensinto the intestinal milieu where they are harmlessly flushed from theintestines by normal metabolic processes.

In one embodiment of the present invention the probiotic organism of thepresent invention was isolated from a traditional fermented yogurt-likeAsian dairy product. The screening process was limited to traditionalfermented yogurt-like Asian dairy products with at least a ten-yearhistory of safe human consumption. Probiotic bacteria isolation wasperformed using three selective microbiological media using methodsknown to those of ordinary skill in the art of microbiology.Lactobacilli selective media included SL medium supplemented with 0.05%cystein, Bifidobacterium spp. were selected for using trypticase peptoneyeast extract medium containing antibiotics; and for Streptococcus spp.were isolated using trypticase yeast extract cystein medium.

Candidate probiotic lactobacilli were be catalase negative, glucosehomofermentative, Gram-positive non-spore forming rods demonstrating lowpH, gastric acid and bile resistance. The lactobacilli isolates'inability to grow at pH 9.0 coupled with their ability to grow onacetate containing media served to distinguish them from Carnobacteriumspp. A total of 81 isolates were classified as candidate probioticlactobacilli based on these criteria and were further characterized withrespect to the following criteria: i) resistance to low pancreaticjuice; ii) adherence ability to sub-epithelial matrices such asBiocoat™, Matrigel™ (Becton Dickinson, Bedford, Mass.) and to culturedintestinal epithelial cells (Caco-2 cell line); iii) their ability tocompetitively exclude enterohemorrhagic E. coli serotype O157:H7adherent to collagen matrices; and iv) their capacity to reduce ammoniaand sulfide containing compounds.

After analyzing all 81-candidate probiotic lactobacilli, two strainswere identified having all of the above-identified characteristics.These strains were designated strain KE97 and strain KE99 (re-designatedKE01). Finally the growth-multiplication rate (generation time asdetermined by impedance detection using BioMerieux™ Bactometer System),stability of strains in continuous culture, freeze-drying and revivalcharacteristics, and aroma/flavor profiles were ascertained for eachstrain.

The isolated Lactobacillus casei strain KE01 organism is maintained in asubstantially pure culture for use in preparing probiotic compositionsof the present invention. As used herein “substantially pure culture”refers to a bacteriological culture that results in only oneidentifiable organism when cultured on solid or semi-solidmicrobiological culture media. It is understood that extremely lowlevels of cells from other bacterial species may be present; however,these cells are either non-viable, non-cultivable or below the thresholdof detection using classical, non-genome-based, microbiologicaltechniques. The term “non-genome-based” is intended to excluded suchmethods as PCR detection or similar methods used to detect microbial DNAor RNA.

Real-Time PCR (RT-PCR) for Specific Measurement of Strain KE01 forQuality (Purity) and Quantity (Total Bacterial Numbers)

In addition to estimating CFU counts by standard plate counts on MRSagar, RT-PCR assay was used for qualitative/quantitative measurement ofsurvival and revival rates of strain KE10 in oil-based formulations.Primers specific for strain KE01 were developed and the RT-PCR wasperformed as described below:

Primer Design

Primers for the amplification of species-specific Lactobacillus caseiKE01 (ATCC-PTA3945) set were synthesized (Table 1). All primers weresynthesized using the standard desalting processes (Integrated DNATechnologies, Coralville, Iowa). The primer set for strain KE01 wasdesigned by amplification of the 16S rRNA gene of Lactobacillus spp.,sequenced (City of Hope Research Center, Duarte, Calif.) and comparedfor base additions, deletions, substitutions, etc. against a public genebank using Lasergene (DNAStar, Madison, Wis.) software. A primer set forReal-Time PCR was then developed specific for the KE01 strain. Thisprimer set creates a 154 by fragment. All primer sets were rehydrated to100 mM with molecular grade water (Eppendorf, Hamburg, Germany) andstored at −20° C. until used.

TABLE 1 Name Primer Sequence KE01 F1 5′-TTG TCA CCG GCA GTT CTT AC-3′(SEQ ID NO 1) lac upp 5′-TGT CGT CAG CTC GTG TCG T-3′ (SEQ ID NO 2)

Real-Time PCR

Quantitative real-time PCR on the iCycler iQ was performed in duplicateon 7 μl of template DNA per 25 μl reaction. The iQ supermix reactionsconsisted of iQ supermix (Bio-Rad, Hercules, Calif.) at a finalconcentration of 1×, 10 nM fluorescein, SYBR Green I with the supermixmaster mix (50 mM KCl, 20 mM Tris-HCl, 0.2 mM dNTP, 25 units/ml iTaq DNAPolymerase, 3 mM MgCl₂).

Reactions were amplified in a 96-well thin wall PCR plate (Bio-Rad)using the following parameters: 95° C. for 3 min, followed by 40 cyclesof denaturation at 95° C. for 10 sec, annealing at 66° C. for 15 sec.Melt curve analysis was performed immediately following amplification byramping the temperature from 55° C. to 95° C. The presence of a singlePCR product was verified both by the presence of a single meltingtemperature peak representing a specific product (vs. a nonspecificprimer-dimer peak) using iCycler iQ analysis software and by detectionof a single band of the expected size on a 12.5% TBE-polyacrylamide gel.

A standard curve was produced and used to determine the concentrationsof the samples. The concentration values were 0.16 ng, 0.016 ng, 0.0016ng, and 0.00016 ng and used from stock known bacteria (strain KE01). Thestandard concentration was used from stock E. coli (ATCC 43895), andstrain KE01. For total lactobacilli estimations a mixture of strains,i.e. L. casei (ATCC393), L. pentosus (ATCC 8041), L. plantarum (ATCC14917), L. paracasei subsp. paracasei (ATCC 25302) and strain KE01 wereused. The DNA of all stock bacteria were extracted and the DNAconcentrations were determined by the Picogreen® Quantification kit anddiluted accordingly in 1× TE buffer to the appropriate concentrationssuitable for RT-PCR analysis. FIG. 3 depicts the results of real-timePCR standards for strain KE01. Each standard concentration (0.16, 0.016,0.0016, 0.00016 and 0 ng) was run in duplicate (two lines per standard).

DNA Fingerprinting by Random Amplified Polymorphic DNA (RAPD) Assay

The RAPD protocol uses PCR for generating a unique fingerprint forbacterial identification. The analysis by PCR can be performed in arapid and reliable manner. Accordingly, the RAPD assay has been used formolecular identification and finger printing of strain KE01. A total of12 Lactobacillus spp. type strains from the ATCC collection werefingerprinted and compared with the KE01. For the DNA fingerprinting allthe lactobacillus strains were cultivated overnight in MRS broth(Difco). The Lactobacillus strains analyzed for DNA fingerprint arelisted in Table 2.

TABLE 2 Lactobacillus spp. SOURCE Lactobacillus strain KE01 en-N-tech,Inc., California, USA Lactobacillus acidophilus ATCC 4356 Human [L 917;IFO 13951; NCIB 8690] Lactobacillus amylovorus ATCC 33620 Cattlewaste-corn silage Lactobacillus brevis ATCC 14869 Human fecesLactobacillus casei subsp. casei ATCC 393 Cheese [IAM 12473; OrlandL-323] Lactobacillus casei subsp. rhamnosus ATCC [BUCSAV 227; P. A.Hansen 300; NCDO 243; 7469 NCIB 6375; NCTC 6375; NRC 488] Lactobacillusdelbrueckii subsp. lactis ATCC Swiss cheese [DSM 20072; IAM 12476; 12315NCDO 1438] Lactobacillus fermentum ATCC 14931 Fermented beets [NCIB11840] Lactobacillus helvaticus ATCC 15009 Swiss cheese Lactobacillusparacasei subsp. paracasei [NCDO 151; R094] ATCC 25302 Lactobacilluspentosus ATCC 8041 [DSM 20314; NCDO 363; NCIB 8026] Lactobacillusplantarum ATCC 14917 Pickled cabbage [IAM 124771] Lactobacillus reuteriATCC 23272 Human feces

DNA Extraction Method

DNA was extracted from the lactobacilli using the Wizard Genomic DNAPurification Kit (Promega, Madison, Wis.). Briefly, 1 mL of 24-h grownMRS broth culture of each Lactobacillus spp. was harvested bycentrifugation, cells were resuspended in 50 mM EDTA and treated with 10mg/mL lysozyme (Sigma, St. Louis, Mo.) at 37° C. for 60 min.Lactobacilli cells were pelleted by centrifugation and supernatant wasremoved. The bacterial pellets were resuspended in the nuclei lysissolution and incubated at 80° C. for 5 minutes. Cell suspension wasallowed to cool to room temperature and RNAse was mixed into thesolution. The suspension was incubated at 37° C. for 60 min. Afterincubation, protein precipitation solution was added to the mixture.Solution was mixed on vortex and incubated on ice for 5 min. The mixturewas centrifuged for 3 min at 15K×g, supernatant was transferred to afresh tube and the DNA was precipitated with isopropyl alcohol. The DNAwas centrifuged and the isopropyl alcohol was aspirated. The DNA pelletwas washed with 70% ethanol and harvested by centrifugation. Ethanol wasremoved and the pellet was allowed to dry. The DNA was resuspended intris-EDTA buffer.

PCR Amplification of Extracted DNA

One microliter of the extracted DNA was used in the PCR reactions, whichwere carried out on the iCycler (Bio-Rad) using a single arbitrarynucleotide sequence according to Cocconcelli, et al. [Cocconcelli, P Set al., Development of RAPD protocol for typing of strains of lacticacid bacteria and entercocci. Lett. Appl. Microbiol. 21:376-9, 1995]. A2× PCR solution-Premix Taq (Takara, Shiga, Japan) was used for eachreaction. Each reaction contained a total volume of 50 μL, 1.25 units ofTakara Ex Taq DNA Polymerase, 1× Buffer, 200 μM dNTP Mix (2.5 mM each).Final concentration of the primer was 4 μM, and the primer used for theamplification was 5′-AgCAgCgTgg-3′ (Operon Technologies, Inc.,Huntsville, Ala.). The reaction mixtures with the template DNA werecycled through the following temperature profile: 1 cycle of 94° C. for5 min; 40 cycles of 94° C. for 1 min; 29° C. for 1 min; ramp to 72° C.1.5 min and held at 72° C. for 1.5 min; 1 cycle of 72° C. for 2 min; andheld at 4° C.

Gel Electrophoresis

Aliquots of each RAPD amplified reaction (10 μL) were analyzed by 1%(wt/vol) agarose gel electrophoresis in Tris-borate-EDTA bufferaccording to Sambrook et al. [Sambrook J et al., Molecular Cloning—ALaboratory Manual, 2nd Edition. Cold Spring Harbor Laboratory Press, NY,1989]. Gels were run for 2 hr at 120V without cooling. The DNA molecularweight marker Hyperladder I (Bioline, Randolph, Mass.) was used as thestandard. After electrophoresis the gel was stained with ethidiumbromide (5 μg/mL) for 10 min, washed for 5 min and visualized andanalysed on a Fluor-S MultiImager (BioRad).

The RAPD assay using a single 10-mer primer produced distinct bandingpatterns of variable intensities and numbers of amplified products on 1%agarose gel with DNA samples of various lactobacillus reference strainsand strain KE01. Comparison of the different species fragments on thegel to the reference Lactobacillus spp. was noted. The banding patternwith documentation as depicted in FIG. 1 and TABLE 3 serves to uniquelyidentify strain KE01 and provided a genomic fingerprinting library.Based on the genomic fingerprinting a dendogram was deduced as shown inFIG. 2. Ward's Cluster Method of Phylogenic Analysis was applied. Thismethod minimizes the Sum of Squares of any two clusters that can beformed at each step, creating clusters of small sizes. Based on thephylogenic analysis, strain KE01 showed 13% homology with Lactobacillushelvaticus ATCC15009 and 55% homology with Lactobacillus casei ssp.rhamnosus ATCC7469.

A pure culture of Lactobacillus casei strain KE01 was deposited with theAmerican Type Culture Collection, Bethesda, Md., which was assigned thenumber ATCC PTA 3945.

The present inventors have demonstrated that by suspending lactic acidbacilli in edible oils, probiotic/prebiotic compositions result havinggreater viability than previously possible. However, as will be apparentfrom the examples that follow, merely because a particular oil isedible, and in fact may have health giving qualities of its own, it doesnot necessarily follow that all edible oils are satisfactory prebiotics.As can be seen in Table 4 below, the percent recovery versus the controlranged from approximately 59% to over 370% during a four week storage.Based on these observation, the present inventors concluded that fishoil, olive oil, rice-bran oil, and soybean oil demonstrate prebioticeffects where the other oils tested were either not prebiotic, or werein fact antibacterial.

TABLE 3 Lane Band Mol. Wt. Number Number (bp) Identification 2 14661.359 Strain KE-01 2 2 2986.357 2 3 2457.454 2 4 2255.342 2 51565.537 2 6 1354.07 2 7 991.287 2 8 904.264 2 9 596.721 3 1 2457.454Lactobacillus acidophilus 3 2 1231.998 ATCC# 4356 3 3 1003.944 3 4904.264 3 5 861.774 3 6 617.813 4 1 4721.177 Lactobacillus amylovorus 42 1969.14 ATCC# 33620 4 3 1894.057 4 4 1725.33 4 5 1672.497 4 6 1271.4144 7 1203.24 4 8 1023.9 5 1 3611.082 Lactobacillus brevis 5 2 3259.978ATCC# 14869 5 3 2534.42 5 4 2087.693 5 5 1864.831 5 6 1271.414 5 71031.994 5 8 940.583 5 9 888.576 5 10 651.854 6 1 2545.999 Lactobacillusdelbrueckii 6 2 1281.464 ssp. lactis ATCC# 12315 6 3 1036.064 6 4940.583 6 5 892.472 6 6 645.528 7 1 7490.875 Lactobacillus fermentum 7 22545.999 ATCC# 14931 7 3 1732.051 7 4 1281.464 7 5 1036.064 7 6 953.0117 7 900.317 7 8 655.041 8 14117.898 Lactobacillus helveticus 8 4936.649ATCC# 15009 8 3634.241 8 2776.318 8 2394.989 8 2105.687 8 1529.442 81036.064 9 1 3588.07 Lactobacillus paracasei 9 2 1271.414 ssp. paracasei9 3 1107.771 ATCC#25302 9 4 1027.939 9 5 932.388 9 6 892.472 9 7 636.15410 1 6200.546 Lactobacillus plantarum 10 2 4968.224 ATCC# 14917 10 34263.326 10 4 1969.14 10 5 1685.552 10 6 1322.462 10 7 1231.998 10 81027.939 10 9 223.284 11 1 2592.843 Lactobacillus casei ssp. 11 22324.117 rhamnosus ATCC#7469 11 3 1685.552 11 4 1430.79 11 5 1312.091 116 1212.751 11 7 1011.88 11 8 780.732 12 1 9035.455 Lactobacilluspentosus 12 2 6787.29 ATCC# 8041 12 3 2405.288 12 4 2198.014 12 51453.497 12 6 1261.443 12 7 1198.513 12 8 982.651 12 9 667.942 13 12945.799 Lactobacillus casei ssp. 13 2 2374.522 casei ATCC# 393 13 32169.899 13 4 1436.433 13 5 1217.534 13 6 974.089 13 7 876.989 13 8839.445 13 9 661.46 13 10 583.785 14 1 2324.117 Lactobacillus reuteri 142 2114.743 ATCC# 23272 14 3 1402.903 14 4 1184.442 14 5 944.708 14 6832.131 14 7 639.264

As discussed above, the present inventors have discovered that selectingthe proper prebiotic oil provides a means for preserving and enrichingthe viability and hence probiotic activity of the Lactobacillipreparations of the present invention. (Note, non-viable Lactobacillimay also possess probiotic effects, however, non-viable preparations maynot benefit significantly from the prebiotic formulations of the presentinvention.) However, when the optimum prebiotic oil is combined with anoptimized delivery system, overall probiotic activity can be increasedand/or preserved over a longer time period.

Probiotic lactobacilli require anaerobic or microaerophilic conditionsfor optimum viability. Though oil immersion provides low water activityand limited oxygen diffusion, any evacuation of oxygen from themicroenvironment would enhance the probiotic preservation. Accordingly,a novel anaerobic encapsulation system usingnitrogen-purge-instant-bonding (NPIB) as depicted in FIG. 5 has beendeveloped to protect the oil-emulsified probiotic formulations of thepresent invention. This process provides an optimal microenvironmentanaerobic/microaerophilic) condition ideal for the probiotic bacteria.As used herein, the term “anaerobic” will mean a low oxygen tensionenvironment and includes a strict anaerobic environment andmicroaerophilic environments. The NPIB system of the present inventionprovides for oxygen displacement simultaneously with sealing the oilemulsified lactobacilli in a hard, two-piece capsule.

Moreover, the NPIB system of the present invention may also be used withother compositions including, but not limited to powders, oil-basedliquids, oil-based suspensions, oil-based pastes, waxes, low-watercontent emulsions and a variety of bioactive compounds including, butnot limited to lactoferrin. Moreover, the choice of capsular material ishighly flexible. For example in one embodiment of the present inventionnatural two piece hard gelatin/vegetable capsules are used, in anotherembodiment soft gelatin/vegetable capsules are used, additional capsulecompositions included assorted synthetic and natural polymers. The NPIBsystem of the present invention can be incorporated into any type ofcapsule filling equipment, including the manual, semi or fully automatedas well as continuous or intermittent motion devices.

FIG. 5 depicts the process for making the anaerobic encapsulation systemof the present invention. After separation of cap and body (504 and 506respectively in FIG. 5), the oil-based bioactive composition of thepresent invention is added to the capsule body 506. Next, an anaerobicgas such as but not limited nitrogen or argon is injected into thecapsule cap 504 and a sealing solution is applied to the inner, lowersection of the cap (seam 502). This results in instant bonding of thecap 504 and body 506 at the time when cap and body are joined during theclosing step. In one embodiment the sealing solution is anaqueous-alcohol solution comprising from approximately 50% to 99%ethanol, 90% being preferred (for vegetable capsules) or 10% to 70%isopropyl alcohol (IPA), 60% being preferred (for gelatin capsules) and15%-30% IPA being optimum for vegetable-based capsules. However, thoseskilled in the art of formulation and filling technologies understandthat other organic solvents are suitable and combinations of differentsolvents may be used. For example, and not intended as a limitation,vegetable capsules comprising plant-derived celluloses are soluble atvarying degrees in ethanol, isopropyl alcohol or water. The specificratio of these three solvents in a sealant is dependant on severalfactors such as (i) rate of sealant application, (ii) volume of sealantapplied, (iii) method of sealant application, (iv) degree of sealantatomization, (v) method of atomization, and (vi) residence time. Thesefactors are also strictly dependant on the wet-ability, solubility andsoftening properties of the capsular materials. Accordingly, thefollowing conditions should be controlled during the sealing process:(a) the softening should not exceed the structural rigidity of the capduring the closing step. Beyond a certain limit, the cap deforms andfails to hold to the body; (b) the wet-ability and solubility propertiesof the cap should be retained and effectively transferred to the body.This would allow proper fusion of the inner surface of the cap with theouter surface of the body during the closing step; (c) The cap and headshould have excellent closure compatibility, since ethanol and isopropylalcohol evaporate rapidly, while water evaporates slowly over a periodof few hours; (d) the wet-ability and solubility properties are highlycritical during ‘instant bonding’ process.

In one embodiment of the present invention the nitrogen purge andsealing steps are done while the capsule is in a vertical position FIG.5. This differs from prior art procedures that fill and close thecapsules in the vertical position and then immediately ejected thefilled capsule into the horizontal position. This changed orientation(from vertical to horizontal) causes the capsule's fluid contentsmigrate into capsule seam 502 between the capsule halves 504 and 506.Once the capsule seam is contaminated with fluid, the sealing step isconsiderably less efficient resulting in leakage of product from thecapsule and contamination of the capsule contents with air.

The NPIB system of the present invention effectively solves this problemby sealing the capsule immediately after filling and purging before anychange in capsule potion is initiated. Consequently, the capsule seamremains uncontaminated and the seal integrity uncompromised.

In one embodiment of the present invention a method for making aprobiotic composition is provided comprising emulsifying a Lactic AcidBacteria (LAB) in an edible oil, the edible oil having prebioticproperties. Next a hard capsule body 506 is filled with the emulsifiedprobiotic while the capsule is in the vertical orientation. The filledcapsule body 506 remaining in the vertical orientation. Next the cap 504is purged with a noble gas such as, but not limited to nitrogen or argonand a sealing solution is applied to the cap seam 502. Followingapplication for the sealant to the cap seam 502, the cap 504 and capsulebody 506 are brought together and instantly bonded. In some embodimentsbone-dry carbon dioxide gas may be used as well. Finally the filled,purged capsule is sealed with an aqueous-organic solvent solution. It isunderstood by those skilled in the art of formulation and filling thatthe purging process can be done contemporaneously with the fillingprocess.

The following examples are meant to be non-limiting in their scope. Tothe extent specific ranges, compositions, ingredients and conditions arecalled out these are to be considered merely exemplary for the purposesof teaching preferred embodiments and or best mode.

EXAMPLES Example 1 Preparation and Testing of Prebiotic Oils

A pure culture of Lactobacillus casei strain KE01 is revived in afermentation broth media containing proteins, vitamins, minerals andcarbohydrate source. A seed culture is prepared in a fermentor attachedto fermentation vessel. Microbial purity is monitored at defined timepoints (through log phase and end cycle) during the fermentationprocess. The microbial mass is harvested in a sanitized separator andthe slurry of cell concentrate was freeze-dried after mixing withcarriers and cryoprotectants. The freeze-dried lactobacillus cellconcentrate was milled to fine powder using sanitized milling equipment.The quality of lactobacilli powder was assured for purity and viabilityprior to use. The viability was about 10¹⁰ CFU lactobacilli/gm ofpowder.

The probiotic lactobacilli powder is then thoroughly mixed withdifferent oils listed in Table 4 to a final 0.1% emulsion (about 0.1 gpowder containing 10⁹ CFU lactobacilli mixed in 1 mL oil). In oneembodiment of the present invention the emulsion is prepared in 10 mLsterile glass tubes and homogenized in a vortex blender. The blendedmixture is then and sealed with air tights caps before testing. Multipletubes for each type of oil emulsion were prepared, and the tubes werestored at room temperature (28° C.). Tubes containing 10⁹ CFUlactobacilli powder without any oil served as growth controls.

At weekly intervals for four weeks a 1 mL sample of each oil containingL. casei was tested for viability as follows: nine mL of sterile deMannRogosa Sharpe (MRS) broth was added to each test aliquot and thecontents were homogenized using a vortex blender. Serial ten-folddilutions of the homogenized mixture were prepared in MRS broth tubes.All tubes were incubated at 37° C. for 18 hours. Lactobacilli growth wasmeasured as change in optical density at 600 nm at the lowest dilution.The growth of control tubes, measured as optical density at 600 nm atsimilar dilution, was considered 100%. The test results were expressedas percent viability as compared to the Controls (Table 4).

Lactobacilli acclimatized to oil environment within the first weekfollowing emulsification. In subsequent weeks, however, the viability oflactobacilli gradually diminished in certain oils i.e. hazelnut,primrose, pumpkin and terila, suggesting a low probiotic recovery. Incontrast, fish oil (omega), olive oil (extra-virgin), rice-bran oil, andsoybean oil all provided an excellent recovery of lactobacilli.Moreover, based on the results expressed in Table 4 below, the presentinventors have reasoned that flax seed oil and vitamin E oil may alsodemonstrate similar prebiotic activity.

TABLE 4 Recovery of Lactobacilli from selected oils in MRS broth(37.degree. C.). Oil-emulsion Type 1^(st) week 2^(nd) week 3^(rd) week4^(th) week Fish (Omega) 122% 220% 255% 280% Hazelnut 92% 88% 67% 59%Olive (Extra virgin) 195% 314% 379% 374% Primrose 87% 87% 94% 95%Pumpkin 79% 78% 82% 85% Rice-bran 108% 212% 223% 267% Soybean 112% 217%274% 290% Terila (pure) 96% 97% 88% 77% Note: Controls i.e. Lactobacillistored under similar conditions and when revived in MRS considered as100% when compared to the test samples.

Example 2 Preparation of Probiotic Samples in Nitrogen Purged InstantlyBonded Capsules and Testing

Each gelatin/vegetable capsule prepared by the NPIB system containedabout 550 mg emulsion comprising of 10⁹ CFU of lactobacilli and 0.5 mlof fish oil (Omega), olive, rice-bran or soybean oil. Ten capsules ofeach oil-emulsified lactobacilli were kept in several glass storagebottles with air-tight caps and stored at room (28° C.) or refrigerated(4° C.) temperatures. Bottles of each oil-emulsion type were taken fromboth storage conditions, periodically, each month, and the capsularcontents were tested for viability of lactobacilli (colony formingunits, CFU). Capsules made with NPIB system containing 10⁹ CFUlactobacilli without any oil served as controls.

Each gelatin/vegetable capsule was allowed to dissolve in test tubescontaining 10 mL MRS broth for about 30 minutes at room temperature.After release of the capsular contents, tubes were thoroughlyhomogenized using a vortex blender. Serial 10 fold dilutions of thehomogenized mixture were prepared in MRS broth tubes. All tubes wereincubated at 37° C. for 18 h. The growth of control tubes, measured asoptical density at 600 nm at similar dilutions, was considered 100%.Real-time PCR was also used to determine the purity of the revivedstrain KE01 from the encapsulated oil-mixtures and the totallactobacillus mass. The test results were expressed as % viability ascompared to the Controls (Table 5).

All four types of oil-emulsified probiotic lactobacilli demonstrated asignificant recovery rate ranging from 400% to 760% over a period oftwelve-month storage at room temperature. These data compared to the100% revival rate of control lactobacilli suggested a potent prebioticeffect for these oil-emulsion types.

TABLE 5 Percent viability of oil-emulsified lactobacilli (in capsulesprepared by the NPIB system) stored at room (28° C.) temperatures formonths and revived in MRS broth (37° C.) Stored at room temp. (28° C.)Oil-emulsion Type 2^(nd) 3^(rd) 4^(th) (NPIB-System) month month month5^(th) month 6^(th) month Fish (Omega) 565% 568% 572% 525% 482% Olive(Extra-virgin) 735% 763% 759% 683% 403% Rice-bran 484% 439% 448% 382%267% Soybean 411% 409% 401% 289% 230% Note: Controls i.e. Lactobacilliencapsulated using NPIB system, stored under similar conditions and whenrevived in MRS considered as 100% compared to the test samples.

Furthermore, the KE01 cells revived from encapsulated oil-mixtures weretested for purity and probiotic activity compared to their stockcultures. Lactobacilli revived from the oil-mixtures showed a highdegree of purity, with a 100% identity match with strain KE01 accordingto real-time PCR analysis. These revived lactobacilli demonstratedprobiotic profiles, i.e. ammonia/sulfide reduction in vitro, intestinalCaco-2 cell adhesion, and competitive exclusion of enteric pathogensfrom Caco-2 monolayers, similar to their KE01 stock culture. These datasuggested that encapsulation of KE01 in specified oil mixtures with NPIBsystem did not affect or alter the probiotic activity of thislactobacillus upon revival over extend periods of time.

Storage of oil-emulsified probiotic formulations at room temperature isuser-friendly and commercially preferred. Such storage condition couldfree the probiotic product from any refrigeration and withoutcompromising viability of the probiotic. Furthermore, the prebioticenhancement of cellular revival suggested a potent functional activityof the oil-emulsified probiotic encapsulations.

Additional variations considered within the scope of the presentinvention include the addition of viscosity enhancers to theencapsulated contents and extra-capsular coatings. For example,viscosity enhancers such as glycerols (eg. glycerine); glycols (e.g.,polyethylene glycols, propylene glycols); plant-derived waxes (e.g.,carnauba, rice, candililla), non-plant waxes (beeswax); emulsifiers(e.g., lecithin); and silicas (e.g., silicon dioxide) are compatibleingredients for NPIB technology. These viscosity enhancers could provideuniform dispersion of the capsular contents and also could improvemicroaerophilic/anaerobic conditions for the probiotic organism in theencapsulation.

Extra-capsular coatings serve various applications including tastemasking, delayed release, vapor/gas diffusion barrier, flavor, color,aroma, enteric protection and leak safety. Such coatings include but arenot limited to celluloses (e.g., methyl- and ethyl-celluloses);methacrylates; and shellacs (e.g., pharmaceutical glazes), just to namea few.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

The terms “a” and “an” and “the” and similar referents used in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein are merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is hereindeemed to contain the group as modified thus fulfilling the writtendescription of all Markush groups used in the appended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on those preferred embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above citedreferences and printed publications are herein individually incorporatedby reference.

I claim:
 1. A probiotic composition comprising: a Lactic Acid Bacteria(LAB) suspended in an edible oil wherein said edible oil has prebioticproperties
 2. The probiotic composition according to claim 1 whereinsaid LAB is selected from the group consisting of Lactobacillusacidophilus, L. amylovorus, L. brevis, L. bulgaricus, L. casei spp.casei, L. casei spp. rhamnosus, L. crispatus, L. delbrueckii ssp.lactis, L. fermentum, L. helvaticus, L. johnsonii, L. paracasei, L.pentosus, L. plantarum, L. reuteri, L. sake, Bifidobacterium animalis,B. bifidum, B. breve, B. infantis, B. longum, Pediococcus acidilactici,Propionibacterium acidipropionici, P. freudenreichii, P. jensenii, P.theonii, Streptococcus cremoris, S. lactis, S. thermophilus andcombinations thereof.
 3. The probiotic composition according to claim 1wherein said prebiotic edible oil is selected from the group consistingof fish oil, olive oil, rice-bran oil, soy oil and combinations thereof.4. The probiotic composition according to claim 1 wherein said LAB is L.casei strain KE01 having ATCC number PTA
 3945. 5. A pair of KE01specific primers wherein said KE01 specific primers consists of positiveand negative strand and said positive primer is SEQ ID NO 1 and saidnegative strand is SEQ ID NO 2.